Regulation of substrate activity

ABSTRACT

A method for regulating substrate activity in vivo is useful for the treatment of medical disorders such as inflammation, arteriolosclerosis and angiogenesis. The method involves the administration of an effective amount of a DPP-IV inhibitor to a patient in need of such treatment.

RELATED APPLICATIONS

This application is a U.S. National Stage Application under 35 U.S.C.§371 of PCT International application PCT/US99/18315 designating theUnited States of America, published under PCT Article 21(2) in English,and filed Aug. 13, 1999, which claims the benefit under 35 U.S.C.§119(e) of U.S. provisional application Ser. No. 60/097,376, filed Aug.21, 1998.

BACKGROUND OF THE INVENTION

This invention relates to inhibitors for dipeptidyl peptidase IV whichare used to regulate substrate activity. As used herein, the term“substrate” denotes chemokines, cytokines and biological peptides whichare substrates for DPP-IV. This invention also relates to the use ofDPP-IV inhibitors in the treatment of medical disorders which may resultfrom the inactivation of substrates implicated in the medical disorder.As used herein, dipeptidyl peptidase IV is alternatively described as“DPP-IV,” “DP-IV” and “CD26.” CD26 is an ectoenzyme with activityidentical to that of DPP-IV.

DPP-IV is a serine type exopeptidase with high substrate specificitycleaves N-terminal dipeptides from proteins if the penultimate aminoacid is proline, or in some cases alanine (Fleischer, B. Immunol. Today15:180 (1994)).

A class of low molecular weight synthetic monomeric molecules with highaffinity for CD26 have previously been developed and characterized (G.R. Flentke et al., Inhibition of dipeptidyl aminopeptidase IV (DP-IV) byXaa-boroPro dipeptides and use of these inhibitors to examine the roleof DP-IV in T-cell function, PNAS (USA) 88, 1556–1559 (1991); W. G.Gutheil and W. W. Bachovchin, Separation of L-Pro-DL-boroPro into ItsComponent Diastereomers and Kinetic Analysis of Their Inhibition ofDipeptidyl Peptidase IV: A New Method for the Analysis of Slow,Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). Thesemolecules have been shown to be potent and specific synthetic inhibitorsfor CD26-associated DP-IV proteinase activity.

Representative monomeric structures of thesetransition-state-analog-based inhibitors, Xaa-boroPro, wherein Xaa is anamino acid residue, include Pro-boroPro, Ala-boroPro, Val-boroPro, andLys-boroPro. BoroPro refers to the analog of proline in which thecarboxylate group (COOH) is replaced with a boronyl group [B(OH)₂].Pro-boroPro, the most thoroughly characterized of these inhibitors, hasa Ki of 16 picomolar (pM) (W. G. Gutheil and W. W. Bachovchin, supra).Val-boroPro has an even higher affinity, with a Ki of 1.6 pM (W. G.Gutheil and W. W. Bachovchin, supra; R. J. Snow et al., Studies onProline Boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV:Identification of a Cyclic Species Containing a B—N Bond, J. Am. Chem.Soc. 116, 10860–10869 (1994)). Thus, these Xaa-boroPro inhibitors areabout 10⁺⁶ fold more potent than the next best known inhibitors.

U.S. Pat. No. 4,935,493 (Bachovchin '493) and U.S. Pat. No. 5,462,928(Bachovchin '928), both of which are incorporated herein by reference,disclose protease inhibitors and transition state analogs (the '493patent) and methods for treating transplant rejection in a patient,arthritis, or systemic lupus erythematosis (SLE) by administering apotent inhibitor of the catalytic activity of soluble amino peptidaseactivity of dipeptidyl peptidase type IV (G. R. Flentke et al., supra).

Chemokines, or chemoattractant cytokines, are a family of small proteinswith a conserved cysteine motifs. These small proteins have beenimplicated in a wide range of disease states, such as acute and chronicinflammatory processes, angiogenesis, leukocyte migration, regulation ofcell proliferation and maturation, hematopoiesis, viral replication, andother immunoregulatory functions. Chemokines are expressed by a numberof different cells and have distinct but overlapping cellular targets.

There are two groups of chemokines defined according to their structuralcharacteristics: the CXC and the CC groups. In addition, C-chemokinesand CX3C-chemokines have been identified. Members of the CXC group,which include SDF-1 and IL-8, attract mostly neutrophils, while the CCgroup acts on monocytes and granulocytes. The CC group includes suchmolecules as human monocyte chemotactic protein 1 (MCP-1) and RANTES.MCP-1 and RANTES are potent direct mediators of the release of histamineby human basophils. Both groups of chemokines are involved in lymphocytemigration to inflammatory sites.

The majority of chemokine receptors are transmembrane spanning moleculeswhich belong to the family of G-protein-coupled receptors. Many of thesereceptors couple to guanine nucleotide binding proteins to transmitcellular signals. Chemokines and receptor expression is upregulatedduring inflammatory responses and cellular activation. Chemokines,through binding to their respective receptors, have been shown to beinvolved in a number of physiologic conditions. For instance, chemokinesof the CXC group, like Interleukin-8, can stimulate angiogenesis, whilePlatelet Factor-4, growth-related oncogene-β (GRO-β) and interferon-γinduced Protein-10 (IP-10) inhibit endothelial cell proliferation andangiogenesis. Interleukin-8 stimulates endothelial cell proliferationand chemotaxis in vitro, and appears to be a primary inducer ofmacrophage induced angiogenesis. It was shown that the activities ofthese chemokines are dependent on the NH₂-terminal amino acid sequence(Streiter et al., J. Biol. Chem., 270; pages 27348–27357). SDF-1,another CXC chemokine, is active in the recruitment and mobilization ofhematopoietic cells from the bone marrow, as well as the attraction ofmonocytes and lymphocytes. In addition, it interferes with cellularinfection of HIV-1 by blocking the interaction of HIV-1 with CXCR-4. Aswith other chemokines in this group, the amino terminal sequenceregulates its activity (Shioda, T., et al., PNAS, 95; pages 6331–6336).

Chemokine receptors have been shown to serve not only as receptors tochemokines, but most recently have been identified as receptors for avariety of microbes and the HIV-1 virus. For instance, the Duffy bloodgroup Ag, a chemokine receptor on erythrocytes, is the receptor for themalaria parasite Plasmodium vivax, and the platelet activating receptoris a receptor for Streptococcus pneumonia.

A number of chemokines, such as RANTES, MIP-1 and SDF-1, or cytokineslike IL-2, or peptides like GLP-1, GLP-2, and Substance P, aresubstrates for DPP-IV. DPP-IV cleaves peptides at the NH₂ terminus ifthe penultimate amino acid is proline. Several cytokines and chemokineshave the conserved sequence NH₂X-Pro-X, and have been shown to besubstrates for DPP-IV. DPP-IV is expressed on the surface of T cells andmacrophages. The relationship of CD26 protease activity to its immunefunction is not clear, however there are indications that cleavage byCD26 of the NH₂-dipeptide of several cytokines changes their receptorspecificity and/or their functional activities.

Cytokines that are known to be potential substrates for DPP-IV includeG-CSF, erythropoietin, IL-1β, IL-2, IL-3, IL-6, IL-11, TNF-β and GM-CSF.

In addition to cytokines and chemokines, a number of biologically activepeptides have, on their amino termini, the amino acid sequenceXaa-Pro-Xaa, which serves as the substrate for DPP-IV. Among these arethe Glucagon Like Peptides, GLP-1 and GLP-2. GLP-1 is involved ininsulin release and glucose uptake, and cleavage by DPP-IV causesinactivation of its activity. Inhibition of DPP-IV will result in theprolonged activation state of this peptide, and represents a therapeuticindication of DPP-IV inhibitors. GLP-2 peptide is involved in intestinalgrowth and nutrient uptake, and increased activity of GLP-2 will resultin an increased nutrient uptake for individuals with intestinaldiseases.

In addition to the foregoing, CD26 is known to be highly expressed onhepatosplenic T cell lymphoma, and DPP-IV activity, or the ability ofCD26 to bind to collagen fibronectin on the extracellular matrix, may bepart of the pathogenic mechanism utilized in by neoplastic cells (Ruizet al., Cytometry 1998, 34: pages 30–35).

DPP-IV and DPP-II levels are known to be significantly increased in thegingival crevicular fluid of patients with periodontitis. Increasedlevels of these two proteases seem to be associated with increasedattachment loss. It is believed that collagen-CD26 interactions are apart of the pathological observations.

PCT published application WO 95/11689 discloses the use of inhibitors ofDPP-IV to block CD26, thereby blocking entry of HIV into CD26-bearingcells. These inhibitors are tetrapeptides having the general formulaX-Pro-Y-boroPro where X and Y are chosen from any amino acid. Althoughthe dipeptides are also disclosed, the reference states that dipeptidesare unstable, and tetrapeptides are preferred. The inhibitors are usedto treat pre-symptomatic HIV-infected patients not by neutralizing thevirus, but by blocking viral entry into the cells. The reference doesnot disclose the effect of DPP-IV inhibition on chemokine activity.

SUMMARY OF THE INVENTION

According to the present invention, a method is provided for thetreatment of a medical disorder in a patient which is mediated bysubstrate activity. Pursuant to this method, a pharmaceuticalcomposition is administered to the patient in an amount which iseffective to inhibit DPP-IV activity. This pharmaceutical compositioncontains, as an active ingredient, a compound represented by the generalformula PR, where P is a targeting moiety that binds to DPP-IV, and R isa reactive group that reacts with a functional group in DPP-IV,preferably a reactive center of DPP-IV. Preferably, the activeingredient is a compound comprising an amino group covalently bonded toan alpha-amino boronic acid analog of proline (the term “boroPro” isused herein to designate such an analog having the carboxyl group ofproline replaced with a B(OH)₂ group, where (OH)₂ represents twohydroxyl groups and B represents boron). The active ingredient cantherefor be designated as Xaa-boroPro, where Xaa is an amino acidresidue. Most preferably, the active ingredient is Val-boroPro whereinthe carboxy terminal boroProline is coupled via a peptide linkage inaccordance with standard peptide chemistry to a valine amino acidresidue.

This active ingredient acts to suppress DPP-IV activity for thosesubstrates which are DPP-IV substrates, resulting in an increase inbioavailability of active chemokine in vivo. The net effect is apositive therapeutic benefit to the patient, particularly with respectto certain disease states, such as inflammation, angiogenesis,arteriolosclerosis, intestinal diseases, diabetes, anorexia, andanti-tumor activity, which could not have been predicted on the basis ofcurrent knowledge of DPP-IV activity or inhibition.

Substrates which are applicable to the method of this invention includethose which share a conserved NH₂-X-Pro sequence (where X is any aminoacid or a short peptide). These substrates for DPP-IV, and theiractivity is altered upon cleavage of the N-terminal sequence. Thealtered substrates can theoretically possess either enhanced orattenuated activity, but they will most typically be inactivated to someextent. Specific examples of chemokines which are inactivated by DPP-IVinclude, but are not limited to, SDF-1, RANTES, MIP-3. The inhibition ofDPP-IV activity prevents the digestion of chemokines, cytokines andgrowth factors. The substrates of this invention are implicated in avariety of disease states, including inflammation, arteriolosclerosis,angiogenesis, and anti-tumor activity. Inhibition of DPP-IV by theactive compounds of this invention is believed to result in thebioavailability of the substrates for enhancing responses to medicaltrauma.

The pharmaceutical composition will typically include the activecomponent (preferably Xaa-boroPro, and most preferably Val-boroPro), anda pharmaceutically acceptable carrier. The pharmaceutical compositioncan also include various adjuvants, enhancers, cytokines, etc., as areknown to those skilled in the art. Patient dosages will generally bewithin the range of 0.001 mg/kg to 100 mg/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the bioavailability of PT-100 (Val-boroPro), and the HCland methane sulfonate salts of PT-100.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the use of certain compounds to inhibit DPP-IVactivity when appropriately administered to a subject. As used herein,the term “subject” is intended to mean a human, non-human primates,dogs, cats, sheep, goats, horses, cows, pigs and rodents. Preferably,the subject is a human patient undergoing medical treatment.

It has now been discovered that inhibition of DPP-IV activity results inthe alteration of certain substrates which are implicated in variousmedical disorders. Since the substrates of this invention are substratesfor DPP-IV, it is believed that DPP-IV acts on the substrates in vivo tocleave the two N-terminal sequence where the penultimate amino acid isproline. Alternatively, other peptidases such as peptides A or N couldcleave the terminal sequence to expose the Xaa-Pro-Xaa moiety. Thiscleavage may result in altering the receptor specificity orfunctionality of the substrate, and typically will result in changes ofthe activity of the substrate. This may directly effect the patient'sresponse to a particular medical disorder or trauma. For instance,certain chemokines, such as SDF-1, MIP-1 and RANTES, are known to act asattractants for lymphocytes, monocytes, etc. Cleavage of thesechemokines by DPP-IV alters their activity and affects the migration ofthe lymphocytes to the site of an inflammation, hematopoiesis or immunefunction. The DPP-IV inhibitor acts to block DPP-IV from cleaving thechemokine, either through competitive interaction with DPP-IV orattachment to the active site.

Active Compounds

Compounds useful in the invention include, but are not limited to,compounds that inhibit DPP-IV and are embraced by the formula PR,wherein P represents a targeting moiety that binds to DPP-IV and Rrepresents a reactive group that reacts with a functional group inDPP-IV, preferably a reactive center of DPP-IV. P can be any moleculethat binds DPP-IV, including DPP-IV binding molecules embraced by theformula: D˜A₁-A₂-A₃-A₄, wherein D is independently selected from thegroup consisting of NH and NH₂, wherein N represents any isotope ofnitrogen, wherein H represents any isotope of hydrogen; “˜”independently, is selected from the group consisting of a single bondand a double bond; A₁ is selected from the group consisting of a C, a CXand an N, wherein C represents any isotope of carbon, X represents anyatom that forms a single bond with carbon; each A₂, A₃, and A₄,independently, is selected from the group consisting of a CX moiety, aCXZ moiety, a CZ moiety, a NX moiety, and an O, wherein X and Z,independently are selected from the group consisting of any atom thatforms a single bond and any atom that forms a double bond with C or Nand wherein O represent any isotope of oxygen.

Compounds useful according to the invention include the followingalternative structures designated as Group I or Group II.

Group I has the structure:

where H represents a hydrogen; C represents a carbon; O represents anoxygen; N represents a nitrogen; each R, independently, is chosen fromthe group consisting of the R groups of an amino acid, includingproline; X represents any atom that forms a single bond with carbon,including hydrogen and halogens; Y is

and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈, separately is a group whichdoes not significantly interfere with site specific recognition of theinhibitory compound by DPP-IV, and allows a complex to be formed withDPP-IV. Preferably, R₁–R₈ are H each H represents a hydrogen atom; and qand p are integers which are independently varied between 0 and 4inclusive.

Alternatively, Group I has the structure:

where n is between 0 and 3 inclusive,

-   -   each G₂ and G₃ independently is H or C₁–C₃ (one to three carbon        atoms) alkyl;    -   G₁ is NH₃ (H₃ represents three hydrogens);    -   or G₁ is        (H₂ represents two hydrogens),    -   or G₁ is NG₄, where G₄ is        where G₅ and G₆ can be NH, H, or C₁–C₃ alkyl or alkenyl with one        or more carbons substituted with a nitrogen. G₁ bears a charge,        and G₁ and G₂ do not form a covalently bonded ring structure at        pH 7.0.

Group I may also have the structure:

where one or two of the a, b, c, d, e, and f group is N, and the restare C, and each S₁–S₆ independently is H or C₁–C₃ alkyl. Group I mayalso include a five membered unsaturated ring having two nitrogen atoms,e.g., an imidazole ring.

Group II has the structure:

where T is a group of the formula:

where each D₁ and D₂, independently, is a hydroxyl group or a groupwhich is capable of being hydrolyzed to a hydroxyl group in aqueoussolution at physiological pH;

-   -   or T is a group of the formula:        where G is either H, F, or an alkyl group containing 1 to 20        carbon atoms and, optionally, heteroatoms which can be N, S or        O;    -   or T is a phosphonate group of the formula:        where each J, independently, is any number of N, H, C, O or S        atoms, in any combination, or O-alkyl, N-alkyl, or alkyl, each        O-alkyl, N-alkyl or alkyl containing 1–20 carbon atoms and,        optionally, heteroatoms which can be N, S, or O;    -   or T is        or        where R is an alkyl, or aryl group and may be substituted or        unsubstituted, an alphaketo ester; or    -   T is generally able to form a complex with the catalytic side of        a DPP-IV.    -   Y is:        and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈, separately is a        group which does not significantly interfere with site specific        recognition of the inhibitory compound by DPP-IV, and allows a        complex to be formed with DPP-IV. Preferably, R₁–R₈ are H. X is        any number of C, H, O, S, or N atoms, in any combination,        including any amino acid or organic molecule, and m can vary        from 0 to 20.

In preferred embodiments, T is a boronate group, a phosphonate group, acyano group, or a trifluoroalkyl ketone group; each R₁–R₈, is H; each R₁and R₂ is H, and each Y is CH₂—CH₂; each R is independently chosen fromthe R group of proline and alanine; the inhibitory compound has abinding or dissociation constant to DPP-IV of at least 10⁻⁹ M, 10⁻⁸ M oreven 10⁻⁷ M; and each D1 and D2 is, independently, F or D1 and D2together are a ring containing 1 to 20 carbon atoms, and optionallyheteroatoms which can be N, S, or O. These compounds are described inU.S. Pat. No. 5,462,928, hereby incorporated by reference.

The more preferred compounds are of the formula:

where each D₁ and D₂, independently, is a hydroxyl group or a groupwhich is capable of being hydrolyzed to a hydroxyl group in aqueoussolution at physiological pH; and wherein X is a targeting moiety thatmimics the site of a substrate recognized and cleaved by DPP-IV, andpreferably is an amino acid, an imino acid, or a peptide which mimicsthe site of a substrate recognized by a post prolyl cleaving enzyme; Cis bonded to B in the L-configuration; and the bonds between B and C,and between Y and N are peptide bonds. By the expression “C is bonded toB in the L-configuration” is meant that the absolute configuration ofthe C is like that of an L-amino acid.

The active Xaa-boroPro compound of this invention can have an open chain(linear) form or a cyclic form. The linear form can be converted to thecyclic form by a trans to cis isomerization of the proline, and theformation of a new N—B bond. Accordingly, “cyclic form” refers to thecyclized structure of the compounds described in the foregoing formulathat are the boron analogs of diketopiperazine.

In a particularly preferred embodiment of the invention, the activecompound is an Xaa-boroPro compound, and still more preferably is aVal-boroPro compound. A “Val-boroPro compound” refers to a compound asdefined in the formula above in which the carboxy terminal boroPro iscovalently coupled via a peptide linkage in accordance with standardpeptide chemistry to a valine amino acid residue. In a most preferredembodiment, the compound of the invention is Val-boroPro (also referredto by the manufacturer's designation “PT-100”).

The preferred active compounds have targeting moieties that are peptideswhich mimic the substrate binding site of DPP-IV. Peptide analogs andnonpeptides or peptidomimetics also can be used as targeting moieties.Such molecules can be rationally designed based upon the known sequenceof substrates of DPP-IV or can be identified using combinatorialchemistry and screening assays such as are described below.

The development of phage display libraries and chemical combinatoriallibraries permits the selection of synthetic compounds which mimic thesubstrate binding site of a protease such as DPP-IV. Such libraries canbe screened to identify non-naturally occurring putative targetingmoieties by assaying protease cleavage activity in the presence andabsence of the putative phage display library molecule or combinatoriallibrary molecule and determining whether the molecule inhibits cleavageby the protease of its natural substrate or of a substrate analog (e.g.,a chromophoric substrate analog which is easily detectable in aspectrophotometric assay). Those phage library and/or combinatoriallibrary molecules which exhibit inhibition of the protease then can becovalently coupled to the reactive groups R disclosed herein and againtested to determine whether these novel molecules selectively bind tothe protease (e.g., by repeating the above-noted screening assay). Inthis manner, a simple, high-through-put screening assay is provided foridentifying non-naturally occurring targeting moieties of the invention.

Substrates

The substrates of this invention are those substrates which share aconserved NH₂-X-Pro sequence (where X is any amino acid or a shortpeptide) at the NH₂ terminus of the molecule. Substrates having thisstructural configuration act as substrates for DPP-IV. The inhibitoryeffect of the present compounds on DPP-IV serves to increase thebioavailability of the substrate in the subject, which in turn resultsin a positive biological result. For instance, increased bioavailabilityof selected substrates can produce an increase in immune andantiinflammatory function in the subject. Suitable substrates are knownin the art, but until now, no one has correlated the use of DPP-IVinhibitors with a positive medical effect on those conditions which aremediated by the presence and activity of the substrates.

Typical substrates which are the subject of this invention are morefully described below. It should of course be appreciated, however, thatother unspecified substrates having conserved NH₂-X-Pro sequences,although not specifically described, would also be within the scope ofthis invention.

SDF-1, or stromal cell-derived factor 1, is a CXC chemokine containing aproline residue at the second position form the N-terminus of themolecule. SDF-1 acts on lymphocytes and monocytes but not neutrophils invitro, and is an effective and potent mononuclear cell attractant invivo. SDF-1 is expressed in a broad range of tissues, it may assist inthe treatment of arteriolosclerosis, and it also may have anti-HIVactivity.

MIP-1 (macrophage inflammatory protein-1) is an attractant forlymphocytes which are essential for immune and inflammatory responses.

RANTES (regulated on activation, normal T-cell expressed and secreted)modulates integrin adhesion and has also been implicated in inflammatorydiseases.

GLP-1 (glucagon-like peptide-1) is known to stimulate insulin secretion.This effect is limited in vivo due to the rapid degradation of GLP-1 byDPP-IV. DPP-IV inhibition, using the active compounds of this invention,would potentiate its insulinotropic effects, and may provide assist inthe treatment of diabetes.

GLP-2 (glucagon-like peptide-2) is known to stimulate small intestinalgrowth through the induction of intestinal epithelial proliferation.GPL-2 is also inactivated by DPP-IV in vivo. DPP-IV inhibition mayresult in increased capacity for nutritional digestion and absorption invivo, and provide a treatment for AIDS, anemia and anorexia. GLP-2 maybe therapeutically useful to enhance mucosal regeneration in patientswith intestinal diseases. Inhibition of DPP-IV promotes the absorptionof enterostatin and desanginine-enterostatin across rat jejunum.

G-CSF (granulocyte-colony stimulating factor) is a growth factor forhematopoietic cells such as neutrophils.

EPO (erythropoietic-6) is a red blood cell growth factor.

IL-6 (Interleukin-6) is a hematopoietic and lymphocyte growth factor.

IL-11 (Interleukin-11) is a lymphokine.

IL-8 (Interleukin-8) is a hematopoietic growth factor, angiogenesiscytokine.

Substance P is a neuropeptide and a hematopoietic growth factor.

Other suitable chemokines include Substance P, which has vasoactiveproperties, monomeric fibrin, which effects blood clotting, fibronectin,which promotes binding of hepatocytes and could enhance liverregeneration, MIP-3, a chemoattractant for B-cells, and collagen typesI, II, III, and IV, which regulate in part the migration of a number ofeffector cells, including T cells, across the endothelial barrier.

Similarly, other medical disorders which may be treated according to themethod of this invention include allergies, angiogenesis, cardiogenesis,anti-tumor responses, hepatic disease, and organ vascularization.

Substrates not specifically disclosed herein, both known and unknown,which are capable of acting as substrates for DPP-IV, are considered tobe fully within the scope of this invention.

Formulation of Pharmaceutical Composition

The compounds of the invention or compositions thereof can beadministered alone or in combination with one another, or in combinationwith other therapeutic agents. For example, treatment with one or moreof the compounds of the invention can be combined with more traditionaltherapies for treating medical disorders, or combined with othercytokines to enhance treatment success.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts. The pharmaceutical compositions also maycontain, optionally, suitable preservatives, such as: benzalkoniumchloride; chlorobutanol; parabens and thimerosal. Carrier formulationsuitable for oral, subcutaneous, intravenous, intramuscular, etc.,administrations can be found in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa.

Routes of Administration

A variety of administration routes are available for treating a subject.The particular mode of delivery selected will depend, of course, uponthe particular compound selected, the severity of the condition beingtreated and the dosage required for therapeutic efficacy. The methods ofthe invention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. Such modes of administrationinclude oral, rectal, topical, nasal, interdermal, or parenteral routes.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the compound of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, elixir or an emulsion. The oral preparation mayinclude an enteric coating.

As used herein, the term “parenteral” includes subcutaneous,intravenous, intramuscular, or infusion. Compositions suitable forparenteral administration conveniently comprise a sterile aqueouspreparation of the compound, which is preferably isotonic with the bloodof the recipient. This aqueous preparation may be formulated accordingto known methods using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water. Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis. They could, however, bepreferred in emergency situations. Oral administration will be preferredfor prophylactic and other treatment because of the convenience to thepatient as well as the dosing schedule.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. The methods include the step of bringing the compoundsof the invention into association with a carrier which constitutes oneor more accessory ingredients. In general, the compositions are preparedby uniformly and intimately bringing the compounds into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds described above, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono-di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe compound is contained in a form within a matrix such as thosedescribed in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, as usedherein, means that the implant is constructed and arranged to delivertherapeutic levels of the active ingredient for at least 10 days, andpreferably 60 days. Long-term sustained release implants are well-knownto those of ordinary skill in the art and include some of the releasesystems described above.

The compounds described herein are administered in effective amounts. Aneffective amount is a dosage of the compound sufficient to provide amedically desirable result. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated, the severity of the condition, the durationof the treatment, the nature of the concurrent therapy (if any), thespecific route of administration, and like factors within the knowledgeand expertise of the health practitioner. An effective amount forstimulating a desired immune response also can be measured, for example,by determining a change in the immune function in a subject (e.g.,increased B cell response, increased cytotoxic T cell response, or anability to slow, halt, or prevent an infection). An effective amount fortreating an autoimmune disorder or allergic disorder would be thatamount sufficient to lessen or inhibit altogether the immune or allergicresponse associated with the disorder so as to slow or halt thedevelopment of or the progression of the disorder. As used in theclaims, “inhibit” embraces all of the foregoing. Likewise, an effectiveamount for treating an immune system disorder is that amount which canslow or halt altogether the symptoms associated with the immune systemdisorder so as to prevent the disorder, slow its progression, or haltthe progression of the immune system disorder. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment.

Generally, doses of active compounds will be from about 0.001 mg/kg perday to 1000 mg/kg per day. It is expected that doses range of 0.01 to100 mg/kg per day will be suitable, preferably orally and in one orseveral administrations per day. Lower doses will result from otherforms of administration, such as intravenous administration. In theevent that a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. Multiple doses per day are contemplated to achieveappropriate systemic levels of compounds.

All patents, references and other documents that are identified in thispatent application are incorporated in their entirety herein byreference.

The following examples are illustrative only and are not intended tolimit the invention in any way. A particularly preferred compound of theinvention, Val-boroPro (“PT-100”), is used in the examples.

Throughout this application and in particular, in each of the examples,particular embodiments are described and illustrated. It is to beunderstood that any of the reactive groups disclosed herein can besubstituted for the particular reactive groups (e.g., boronyl group) asdescribed in the examples.

EXAMPLE 1 General Synthesis of Active Compounds

Synthesis of the boroPro compounds of this invention are described inBachovchin '493. In general, the preparatory technique involvesstraightforward peptide coupling chemistry. The standard peptidecoupling chemistry methods and procedures used in this invention arereadily available. Examples of books using these methods include, butare not limited to, the following citations incorporated herein byreference: P. D. Bailey, An Introduction to Peptide Chemistry, Ed.: JohnWiley & Sons, 1990; Miklos Bodansky, Peptide Chemistry, A PracticalTextbook, Ed.: Springer-Verlag, 1988; Miklos Bodansky, Principles ofPeptide Synthesis, “Reactivity and Structure Concepts in OrganicChemistry,” Volume 16, Ed.: Springer-Verlag, 1984; and Miklos Bodansky,Principles of Peptide Synthesis, “Reactivity and Structure Concepts inOrganic Chemistry,” Volume 21, Ed.: Springer-Verlag, 1984.

The compounds of the invention can begin with the synthesis of H-boroProas taught in WO 98/00439. Use of H-boroPro is for illustrative purposesonly, and is not intended to limit the scope of this invention.

According to WO 98/00439, H-boroPro was prepared by the synthetic routepreviously developed and described (G. R. Flentke, et al., “Inhibitionof dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides anduse of these inhibitors to examine the role of DP-IV in T-cellfunction,” PNAS (U.S.A.) 88, 1556–1559 (1991); also described in U.S.Pat. No. 5,462,928). Alternatively, H-boroPro may be produced by a newprocedure (Kelly, T. A., et al., “The efficient synthesis and simpleresolution of a proline boronate ester suitable for enzyme inhibitionstudies,” Tetrahedron 49, 1009–1016 (1993)). Both of these syntheticroutes reportedly yield racemic H-boroPro pinanediol.

According to WO 98/00439, stereochemically pure L, L and L, Ddiastereomers of Z-Lys-boroPro were prepared by first resolving racemicH-boroPro through crystallization with optically active blockingprotecting groups ((1S, 2S, 3R, 5S)-+-pinanediol isomer) followed bycoupling the isotopically pure L-boroPro and D-boroPro to thestereochemically pure L isomer of lysine (See U.S. Pat. No. 5,462,928).Alternatively, the L,L and L,D diastereomers of Lys-boroPro wereprepared in high optical purity by coupling racemic H-boroPro by L-Lysand separating the resulting diastereomeric Z-Lys-boroPro-diester intoits component L,D and L,L diastereomers using reverse phase HPLC aspreviously described for diastereomeric Pro-boroPro (W. G. Gutheil andW. W. Bachovchin, “Separation of L-Pro-DL-boroPro into Its ComponentDiastereomers and Kinetic Analysis of Their Inhibition of DipeptidylPeptidase IV. A New Method for the Analysis of Slow, Tight-BindingInhibition,” Biochemistry 32, 8723–8731 (1993)).

EXAMPLE 2 Synthesis of Active Cyclic Compounds

In aqueous solution at all pH values, the inhibitors exist as a slowlyequilibrating mixture of two conformations: an open chain structure(linear boroProline compound) which is inhibitory (active species), anda cyclic structure (cyclic boroProline compound) which is non-inhibitory(inactive species). The open, active, inhibitory chain species isfavored at low pH while the cyclized structure is favored at neutral pH.The reaction is fully reversible: the open chain becomes predominant atlow pH. The open chain to cyclic species reaction involves a trans tocis isomerization of the proline and the formation of a new N—B bond.The cyclized structure is the boron analog of a diketopiperazine, aproduct often seen in peptide chemistry. Cyclization liberates oneequivalent of H+ thereby explaining the requirement for base in thecyclization reaction and acid in the opening reaction. The cyclicstructure is quite stable in aqueous solutions of high pH.

Prolonged incubation at high pH does not lead to the completedisappearance of DPP-IV inhibitory activity for any of the Xaa-boroProcompounds examined. This observation was the first evidence that theactive inhibitor was in a conformation equilibrium with a non-inhibitoryspecies rather than undergoing an irreversible inactivation. The halflife for the reformation of the open chain species from the cyclicstructure is surprisingly low. Thus, it was concluded that the loss ofinhibitory activity in aqueous solution was due to a pH dependentconformational equilibrium rather than a degradation reaction.

The fact that the inhibitory activity does not go to zero for any of theXaa-boroPro inhibitors, even after prolonged incubation, together withthe fact that the reverse reaction, i.e., cyclic to open chain is slow,suggested that it should be possible to measure the equilibrium constantfor the conformation equilibrium by measuring the apparent Ki atequilibrium and comparing it with the true Ki. It has been reported thatthe ratio of [cyclic]: [open] forms, at neutral pH, is 156:1 forPro-boroPro and 11130:1 for Val-boroPro (W. G. Gutheil and W. W.Bachovchin, Separation of L-Pro-DL-boroPro into Its ComponentDiastereomers and Kinetic Analysis of Their Inhibition of DipeptidylPeptidase IV. A New Method for the Analysis of Slow, Tight-BindingInhibition, Biochemistry 32, 8723–8731 (1993)). This means that lessthan 1% Pro-boroPro and less than 0.1% of Val-boroPro exists as the openchain, inhibitory species, at equilibrium at pH 7.0. Nevertheless, underthese conditions the inhibitors behave as though they had Ki's of 2.5 nMand 1.8 nM respectively. This apparent Ki of the “fully inactivated”species is still substantially better than, (˜1000-fold) that of otherinhibitors of DPP-IV thus far reported.

The inventors believe that the cyclic compounds of the invention havethe ability to specifically bind to CD26. Accordingly, the inventorspredict that the biological function of the compounds of the inventioncould be significantly increased (approximately 100–1000 times) byorally administering the cyclic compounds of the invention andpermitting the conformational changes, e.g., linearization, to occur invivo (e.g., under the acidic conditions of the stomach).

Thus, if linearization is necessary, it can be accomplished in vivo andtherefore, therapeutic concentrations in the systemic circulation can begenerated in situ and, accordingly, it is believed that the bioactivityof the compounds of the invention can be increased by approximately100–1000 fold. In addition, it is believed that the cyclic boroProlinecompounds of the invention, in lyophilized or solid form, have improvedshelf life properties, thereby contributing to the further utility ofthe compounds of the invention.

Each of the compounds prepared as described above can be purified tohomogeneity using HPLC and its identity can be confirmed by NMRspectroscopy, amino acid composition, or mass spectroscopy as deemednecessary.

The cyclic compounds of the invention can be converted to linear form byadjusting the pH to an acidic pH (e.g., pH range: 1–3) and the potencyof inhibition of CD26 proteinase activity by the linear boroProlinecompounds can be determined using conventional enzyme analysis (exampleprovided below). In addition, the immunomodulatory effects of thecompounds of the invention are evaluated by in vivo experiments usinganimal models and by in vitro experiments using cell culture methodsthat are believed by those of ordinary skill in the art to be predictiveof an in vivo activity.

EXAMPLE 3 Assessment of Functional Activity

The compounds of the invention have at least the following properties:(I) binding site is the DPP-IV active site; and (ii) exhibitcross-species specificity.

The assays which are used to assess functional activity include: DPP-IVactivity, oral and subcutaneous bioavailability assays, and aredescribed below.

EXAMPLE 4 Measuring Standard DPP-IV Activity

Assays to measure DPP-IV activity can be performed on the compounds ofthe invention. Methods for quantitatively measuring the interaction ofsmall peptidomimetic inhibitors with DPP-IV, as well as for theinteraction of CD26 with larger ligands, e.g., the HIV Tat protein, havebeen developed (W. G. Gutheil and W. W. Bachovchin. Separation ofL-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysisof Their Inhibition of Dipeptidyl Peptidase IV. A New Method for theAnalysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723–8731(1993); Gutheil, W. G., and W., B. W. Kinlsq, A Matlab Program forFitting Kinetics Data with Numerically Integrated Rate Equations and ItsApplication to the Analysis of Slow, Tight Binding Data, AnalyticalBiochemistry 223, 13–20 (1994); Gutheil, W. G., et al., HIV-1 Tat Bindsto DP IV (CD26): A possible Mechanism for Tat's ImmunosuppressiveActivity, Proc. Natl. Acad. Sci. U.S.A. 91, 6594–6598 (1994)). Thesemethods use the chromatogenic substrate Ala-Pro-p-nitroanilide (AppNA)and fluorescent substrate Ala-Pro-7-amino-4-trifluoromethyl coumarin(AP-AFC). AppNA and AP-AFC are commercially available (e.g., EnzymeSystems Products, Dublin, Calif.).

EXAMPLE 5 Measuring Oral Bioavailability

An in vitro assay of serum DPP-IV activity has been developed, which canbe used as a surrogate marker to determine the bioavailability of PT-100in serum following subcutaneous or oral administration in mice. Thisassay is based on the ability of PT-100 to inhibit serum DDP-IV proteaseactivity in a dose dependent manner.

As shown in FIG. 1, mice received PT-100 at indicated doses by gavage.Blood samples are taken two hours post administration and serum DPP-IVactivity is determined. DPP-IV activity is measured in a fluorometricassay using the synthetic substrate 7-amino-4-trifluoromethyl coumarin(AFC)-AlaPro (15). Total inhibition of serum DPP-IV activity is achievedat doses of 2 μg or 5 μg PT-100. Doses below 2 μg result in a dosedependent decrease of DPP-IV inhibition.

While the invention has been described with respect to certainembodiments, it should be appreciated that many modifications andchanges may be made by those of ordinary skill in the art withoutdeparting from the spirit of the invention. It is intended that suchmodifications, changes and equivalents fall within the scope of theappended claims.

1. A method for treating a medical disorder in a subject mediated by thealteration of substrate activity comprising administering to the subjectan effective amount of a compound having the formula

wherein each D₁ and D₂ is a hydroxyl group or a group which is capableof being hydrolyzed to a hydroxyl group in aqueous solution atphysiological pH; wherein X is an amino acid; and wherein C is bonded toB in the L-configuration, said amount being sufficient to preventchemokine alteration by inhibiting DPP-IV activity, and wherein themedical disorder is selected from the group consisting ofarteriolosclerosis and insufficient blood clotting.
 2. The method ofclaim 1 wherein the compound is Val-boroPro.
 3. The method of claim 1wherein the compound is cyclic X-boroPro.
 4. The method of claim 1wherein the substrate is selected from the group consisting of SDF-1,RANTES, MIP-1, MIP-3, GLP-2, G-CSF, EPO, IL-6, IL-11, IL-8, Substance P,fibronectin, and monomeric fibrin.
 5. The method of claim 1 wherein thecompound is given to the subject by oral administration.
 6. The methodof claim 1 wherein the compound is given to the subject by parenteraladministration.
 7. The method of claim 1 wherein the effective amount isin the range of 0.01 mg/kg per day to 100 mg/kg per day.
 8. The methodof claim 1 wherein the compound has a binding or dissociation constantto DPP-IV of at least 10⁻⁹ M.
 9. A method for treating an intestinaldisease consisting of administering to a subject in need thereof aneffective amount of a compound having the formula

wherein each D₁ and D₂ is a hydroxyl group or a group which is capableof being hydrolyzed to a hydroxyl group in aqueous solution atphysiological pH, X is an amino acid, and C is bonded to B in theL-configuration, said amount being sufficient to prevent chemokinealteration by inhibiting DPP-IV activity, and a pharmaceuticallyacceptable carrier, wherein the intestinal disease is not a cancer,tumor or neoplasm.
 10. The method of claim 9 wherein the compound isVal-boroPro.
 11. The method of claim 9 wherein the compound is cyclicX-boroPro.
 12. The method of claim 9 wherein the compound is given tothe subject by oral administration.
 13. The method of claim 9 whereinthe compound is given to the subject by parenteral administration. 14.The method of claim 9 wherein the effective amount is in the range of0.01 mg/kg per day to 100 mg/kg per day.
 15. The method of claim 9wherein the compound has a binding or dissociation constant to DPP-IV ofat least 10⁻⁹ M.